the blood-brain barrier: bottleneck in brain drug development · the blood-brain barrier:...

The Blood-Brain Barrier: Bottleneck in BrainDrug Development

William M. Pardridge

Department of Medicine, UCLA, Los Angeles, California 90024

Summary: The blood-brain barrier (BBB) is formed by thebrain capillary endothelium and excludes from the brain�100% of large-molecule neurotherapeutics and more than98% of all small-molecule drugs. Despite the importance of theBBB to the neurotherapeutics mission, the BBB receives in-sufficient attention in either academic neuroscience or industryprograms. The combination of so little effort in developingsolutions to the BBB problem, and the minimal BBB transportof the majority of all potential CNS drugs, leads predictably tothe present situation in neurotherapeutics, which is that there

are few effective treatments for the majority of CNS disorders.This situation can be reversed by an accelerated effort to de-velop a knowledge base in the fundamental transport propertiesof the BBB, and the molecular and cellular biology of the braincapillary endothelium. This provides the platform for CNS drugdelivery programs, which should be developed in parallel withtraditional CNS drug discovery efforts in the molecular neuro-sciences. Key Words: Blood-brain barrier, endothelium, drugtargeting, biological transport, neurotherapeutics.

INTRODUCTION

The blood-brain barrier (BBB) is the bottleneck inbrain drug development and is the single most importantfactor limiting the future growth of neurotherapeutics.1

The BBB problem is illustrated in Figure 1, which is awhole body autoradiogram of a mouse sacrificed 30 minafter intravenous injection of radiolabeled histamine, asmall molecule of only �100 Da in molecular mass.Histamine readily crosses the porous capillaries perfus-ing all peripheral tissues but is excluded from entry intothe brain or spinal cord by the BBB.The histamine example in Figure 1 refutes a common

misconception that most small molecules readily crossthe BBB. As discussed below, the transport of smallmolecules across the BBB is the exception rather thanthe rule, and 98% of all small molecules do not cross theBBB (FIG. 1). Moreover, all large-molecule products ofbiotechnology, such as monoclonal antibodies (mAbs),recombinant proteins, antisense, or gene therapeutics, donot cross the BBB (FIG. 1). Despite the large number ofpatients with disorders of the CNS and despite the factthat so few large- or small-molecule therapeutics cross

the BBB, there are few pharmaceutical companies in theworld today that have built a BBB drug targeting pro-gram (FIG. 1). However, even if a pharmaceutical com-pany decided to develop a BBB program, there would befew BBB-trained scientists to hire because less than 1%of U.S. academic neuroscience programs emphasizeBBB transport biology.Because most drugs do not cross the BBB, and be-

cause the industry is not providing solutions to the BBBproblem, it is not surprising that most disorders of theCNS could benefit from improved drug therapy (FIG. 2).For a small-molecule drug to cross the BBB in pharma-cologically significant amounts, the molecule must havethe dual molecular characteristics of: 1) molecular massunder a 400- to 500-Da threshold, and 2) high lipidsolubility.1 There are only four categories of CNS dis-orders that consistently respond to such molecules, andthese include affective disorders, chronic pain, and epi-lepsy (FIG. 2). Migraine headache may be a CNS disor-der and could also be included in this category. In con-trast, most CNS disorders such as those listed in Figure2 have few treatment options. Parkinson’s disease pa-tients are given L-dihydroxyphenylalanine (L-DOPA) fordopamine replacement therapy.2 As discussed below inthe section on BBB carrier-mediated transport, L-DOPAis an example of a BBB drug targeting strategy. How-ever, there is no neurotherapeutic that stops the neuro-

Address correspondence and reprint requests to William M.Pardridge, M.D., UCLA Warren Hall, 13-164, 900 Veteran Avenue,Los Angeles, CA 90024. E-mail: [email protected]

NeuroRx�: The Journal of the American Society for Experimental NeuroTherapeutics

Vol. 2, 3–14, January 2005 © The American Society for Experimental NeuroTherapeutics, Inc. 3

degeneration of Parkinson’s disease. Similarly, there isno therapy for other neurodegenerative diseases such asAlzheimer’s disease, Huntington’s disease, and amytro-phic lateral sclerosis (ALS). Patients with multiple scle-rosis (MS) are treated with cytokines that work on theperipheral immune system, but which do not perma-nently stop the progression of MS.3 The human immu-nodeficiency virus (HIV) infects the brain early in the

course of acquired immune deficiency syndrome(AIDS).4 HIV in the periphery has been significantlyreduced with highly active antiretroviral therapy(HAART) comprised of multiple small-molecule thera-peutics. However, HAART drugs such as azidothymi-dine, 3TC, or protease inhibitors are substrates for BBBactive efflux transporters, which are reviewed below, andHAART drugs have minimal penetration into brain pa-renchyma. Consequently, the brain remains a sanctuaryfor HIV in AIDS even with HAART.4,5 Brain cancer,stroke, and brain or spinal cord trauma are all examplesof serious CNS disorders for which there is no effectivedrug therapy. The childhood disorders including autism,lysosomal storage disorders, fragile X syndrome, theataxis, and blindness, are serious disorders where there islittle effective treatment. In many of these cases, the geneunderlying the disease is known, but BBB delivery is therate-limiting problem in gene therapy or enzyme replace-ment therapy, and no therapeutics have been developed.Many of the disorders listed in the right-hand column inFigure 2 could be treated with drugs, enzymes, or genesalready discovered. However, these drugs do not crossthe BBB and cannot enter into brain drug developmentbecause no BBB solutions have been developed by in-dustry. Given the absence of effective BBB drug target-ing technology, CNS drug developers are left with thetraditional approaches to solving the brain drug deliveryproblem: small molecules, trans-cranial brain drug deliv-ery, and BBB disruption. A review of these approaches

FIG. 1. Whole body autoradiogram of an adult mouse sacrificed 30 min after intravenous injection of radiolabeled histamine, a smallmolecule that readily enters all organs of the body, except for the brain and spinal cord.

FIG. 2. A review of the Comprehensive Medicinal Chemistrydatabase shows that, of more than 7000 small-molecule drugs,only 5% treat the CNS, and these drugs only treat four disorders:depression, schizophrenia, chronic pain, and epilepsy.6,7 Thereare few effective small- or large-molecule drugs for the majorityof CNS disorders, with the exception of Parkinson’s disease,e.g., L-DOPA, and multiple sclerosis, e.g., cytokines.

WILLIAM M. PARDRIDGE4

NeuroRx�, Vol. 2, No. 1, 2005

shows that none provide solutions to the BBB problemthat could be practically implemented in large numbersof patients.

SMALL MOLECULES

Most small-molecule drugs do not cross the BBB. Ofover 7000 drugs in the comprehensive medicinal chem-istry (CMC) database, only 5% of all drugs treat theCNS, and these CNS active drugs only treat depression,schizophrenia, and insomnia.6 The average molecularmass of the CNS active drug is 357 Da. In another study,only 12% of drugs were active in the CNS, but only 1%of all drugs were active in the CNS for diseases otherthan affective disorders.7

BBB transport of small molecules is limitedSmall molecules generally cross the BBB in pharmaco-

logically significant amounts if 1) the molecular mass of thedrug is less than 400-500 Da, and 2) the drug forms lessthan 8-10 hydrogen bonds with solvent water.1

The permeation of the drug across the BBB does notincrease in proportion to lipid solubility when the mo-lecular weight of the drug is increased. BBB permeationdecreases 100-fold as the surface area of the drug isincreased from 52 Angstroms2 (e.g., a drug with molec-ular mass of 200 Da) to 105 Angstroms2 (e.g., a drug of450 Da).8 Drug diffusion through a biological membraneis not analogous to drug diffusion through solvent water.In contrast to water, diffusion of a drug through a bio-logical membrane is dependent on the volume of thedrug. The classical Overton rules that relate membranepermeation to solute lipid solubility do not predict themolecular weight threshold effect. As noted by Leib andStein nearly 20 years ago,9 the molecular weight thresh-old effect is best predicted by the “hole-jumping” modelof Trauble,10 which posits that solutes undergo a form ofmolecular “hitch hiking” across a biological membraneby moving through small holes in the membrane formedby kinking of the mobile unsaturated fatty acyl sidechains in the phospholipid bilayer.

Hydrogen bondingBBB permeation decreases exponentially with the ad-

dition of each pair of hydrogen bonds added to the drugstructure.11 It does not matter whether the functionalgroup is a hydrogen bond donor or a hydrogen bondacceptor because each hydrogen bond carries equalweight. Hydrogen bond donor groups such as hydroxylsform two hydrogen bonds because a hydroxyl group actsas both a hydrogen bond donor and hydrogen bond ac-ceptor, whereas a carbonyl group only acts as a hydrogenbond acceptor. Once the total number of hydrogen bondson the drug exceeds a threshold of 8-10, there is minimaltransport of the drug across the BBB in pharmacologi-cally active amounts. Both the hydrogen bonding and the

molecular weight of drugs currently emanating fromCNS drug discovery programs generally are higher thandrugs discovered 20 years ago.7 This is because CNSdrug discovery programs now rely extensively on recep-tor-based high-throughput screening (HTS) programs.HTS-based drug screening invariably selects for drugsthat have higher molecular weights and higher hydrogenbonding because these factors enable higher affinity drugbinding to the target receptor.

HTS-based CNS drug discoveryCurrent CNS drug discovery programs are generally

broken down into four major areas: 1) receptor targetidentification, 2) drug “hit” identification, 3) “lead” iden-tification, and 4) drug lead optimization. After screeningseveral hundred thousand small-molecule drugs with agiven target, several hundred hits may be found, leadingto a score of potential drug leads. The HTS drug leadcompounds must then be optimized with respect to dis-tribution, metabolism, and pharmacokinetics (DMPK).12

However, the drugs generally require so much medicinalchemistry to block polar functional groups that the orig-inal high receptor affinity is lost in an attempt to producea drug with acceptable DMPK properties. The difficultyin using medicinal chemistry to increase the lipid solu-bility of a drug is illustrated by considering that there isnot a single drug currently in CNS clinical practice thatis an example of a water soluble drug that was made lipidsoluble with medicinal chemistry optimization such thatthe drug then became pharmacologically active in thebrain in vivo.

The pharmacokinetic ruleWhen medicinal chemistry is used to increase the lipid

solubility of the drug, this may increase penetrationacross the BBB, but it also increases penetration acrossall biological membranes in vivo. Therefore, the lipidizedform of the drug is rapidly removed from the blood, andin pharmacokinetic terms, the plasma area under theconcentration curve (AUC) is substantially decreased forthe lipidized form of the drug. Drug action in brain is afunction of drug uptake, expressed as percent of injecteddose (ID) per gram brain, and the % ID/g is equallydependent on two factors, the BBB permeability-surfacearea (PS) product and the plasma AUC:

% ID/g � �BBBPS product� � �plasmaAUC�

(Eq. 1)

Although an increase in lipid solubility of the drug mayincrease the BBB PS product, there is a proportionaldecrease in the plasma AUC with lipidization. The in-creased BBB PS product and the decreased plasmidAUC have offsetting effects, which minimizes the in-crease in brain uptake caused by lipidization.1

BBB AND CNS DRUG DEVELOPMENT 5


Medicinal chemistry and brain drug leadoptimizationThe use of medicinal chemistry to increase the lipid

solubility of drug to solve the BBB drug delivery prob-lem is problematical for the reasons listed above. How-ever, a new approach to the use of medicinal chemistry tosolve the BBB drug delivery problem is discussed below.Medicinal chemistry can be used to alter the structure ofa lead drug candidate to make that drug transportable onone of several carrier-mediated transport (CMT) systemswithin the BBB. However, redirection of the use of me-dicinal chemistry to increase the carrier-mediated trans-port of a drug, as oppose to the lipid-mediated transportof the drug, requires knowledge on the structural char-acteristics of a drug that enable CMT across the BBB.Therefore, a knowledge base in BBB CMT must bedeveloped before the use of medicinal chemistry to in-crease drug penetration to the brain via endogenous BBBcarriers.

TRANS-CRANIAL BRAIN DRUG DELIVERY

Trans-cranial brain drug delivery approaches attemptto bypass the BBB using one of three neurosurgical-based delivery approaches: intracerebral implantation,intracerebroventricular (ICV) infusion, and convectionenhanced diffusion (CED). The factor limiting either theintracerebral or ICV infusion approach is that eithermethod relies on diffusion for drug penetration into thebrain from the depot site. Solute diffusion decreases withthe square of the diffusion distance.1 Therefore, the con-centration of drug decreases logarithmically with eachmillimeter of brain tissue that is removed from the in-jection site, in the case of intracerebral implantation, orfrom the ependymal surface of the brain, in the case ofICV infusion. The concentration of a small molecule isdecreased by 90% at a distance of only 0.5 mm from theintracerebral implantation site in rat brain.13 The loga-rithmic decrease in drug concentration from the ependy-mal surface following an ICV infusion was shown in the1970s in adult Rhesus monkeys; after ICV drug injec-tion, the concentration of small molecules in brain pa-renchyma removed only 1-2 mm from the ependymalsurface is only about 1-2% of the concentration in theCSF compartment.14 The limited diffusion of drug froman intracerebral implant is shown in Figure 3, which is anautoradiogram of rat brain taken 2 days after the intra-cerebral implantation of a wafer embedded with radiola-beled NGF.15 The size of the wafer is approximatelyequal to the magnification bar in the figure, which indi-cates that there has been minimal penetration of NGFinto brain parenchyma from the implant site. The limiteddiffusion of BDNF into brain parenchyma following in-jection into a lateral ventricle (LV)16 is shown in Figure3. The BDNF is sequestered by the ependymal surface

but does not significantly diffuse into brain parenchyma.This limited diffusion of BDNF into brain parenchyma isnot due to the fact that BDNF is a cationic protein, as asimilar logarithmic decrease in brain penetration is foundfor any drug following ICV injection.14 This slow rate ofdrug diffusion into brain parenchyma is to be contrastedwith the rapid rate of bulk flow of CSF through theventricular compartments. CSF is then rapidly absorpedinto the peripheral bloodstream at the superior sagittalsinus. The ICV injection of drug should be regarded as aslow intravenous infusion rather than a direct adminis-tration of drug into the brain.17 The rapid rate of cytokinedistribution into blood, but minimal penetration intobrain, following an ICV injection has been demonstratedin adult rhesus monkeys.18

The effective penetration of drug into brain can beincreased to a treatment radius of a few millimeters whenbulk flow is used to deliver drug into brain parenchyma,and this is possible by forcing fluid through the brainwith CED. However, the brain has no lymphatic systemand is not designed for a significant intraparenchymalvolume flow. CED in humans with glioblastoma multi-form causes a preferential flow of the forced fluid alongwhite matter tracts.19 CED in the adult Rhesus monkeybrain with glial-derived neurotrophic factor involved theinfusion of relatively small volumes of�0.1 ml/day overa 4-week period.20 This led to diffuse white matter as-

FIG. 3. Trans-cranial drug delivery to the brain. A: Autoradio-gram of rat brain 48 h after an intracerebral implantation of apolymer carrying radiolabeled NGF.15 The size of the polymerapproximates the magnification bar, indicating the NGF has notsignificantly diffused from the implantation site. B: Autoradio-gram of rat brain 24 h after an intracerebroventricular injection ofBDNF into an LV.16 The BDNF distributes to the ependymalsurface of the ipsilateral LV and the third ventricle (3V), but notinto brain parenchyma. C: Convection enhanced diffusion in theprimate brain forces fluid through the brain tissue. The directionof fluid flow, principally via white matter tracts,19 can be tracedwith immunocytochemistry using an antibody to GFAP, whichshows an astrogliotic reaction in the path of fluid flow.20 The holein the brain left by the catheter is noted by the asterisk. The fluidmoved from the catheter in the putamen (Pu) via the internalcapsule (ic) white matter to the caudate (Cd).



trogliosis, which was visualized by immunocytochemis-try of the autopsy primate brain, and immunostainingwith an antibody to GFAP as shown in Figure 3. Inaddition, there was a microglial response and demyeli-nation around the catheter, with extension of the astro-gliotic reaction from the catheter in the putamen (Pu)through the internal capsule (ic) to the caudate (cd) (FIG.3). These findings of an intense astrogliotic reactionalong white matter tracts after CED in the primate brainraise concerns about the long-term effects of this deliv-ery approach for humans.

BLOOD-BRAIN BARRIER DISRUPTION

In parallel with trans-cranial brain drug delivery strat-egies, there has been a significant effort in deliveringdrugs to the brain with BBB disruption after the intraca-rotid arterial infusion of vasoactive agents such as thoselisted in Table 1. The intracarotid arterial infusion of 2 Mconcentrations of poorly diffusible solutes such as man-nitol causes disruption of the BBB owing to osmoticshrinkage of the endothelial cells.21 This is associatedwith severe vasculopathy22 and chronic neuropathologicchanges in rodent models23 and is also associated withseizures in either animal models24 or humans.25 Plasmaproteins such as albumin are toxic to brain cells,26 andBBB disruption allows for the uptake of plasma into thebrain.

Solvent/adjuvant-mediated BBB disruptionThe BBB, like cell membranes in general, is subject to

solvent-mediated disruption with chemicals such as eth-anol, dimethylsulfoxide (DMSO), or detergents such asSDS, or Tween 80 also known as polysorbate-80.27–30

There are numerous examples in the literature where theperipheral administration of a drug, which normallyshould not cross the BBB, is followed by pharmacolog-ical activity in the brain. Such an observation could arise

because the drug is transported across the BBB via anendogenous transport system. However, an alternativeexplanation is that the drug is injected in a diluent that ismembrane destabilizing, and causes BBB disruption. Of-ten the drug is solubilized in solvents such as ethanol orDMSO, or surfactants such as SDS, a Tween detergent,or other surfactants, such as polyethyleneglycol hydroxystearate. Doses of solvents such as ethanol or DMSO ata level of 1-4 g/kg may cause solvent-mediated disrup-tion of the BBB.27,28 This dose of DMSO or ethanol isgiven to animal models with surprising frequency, par-ticularly small rodent models such as mice, which weighonly 20-30 g. The administration of just 50 �l of 50%DMSO to a 20-g mouse is equivalent to 1.25 g/kgDMSO, and there are examples in the literature of phar-macologic effects achieved in brain following systemicadministration of drugs that normally do not cross theBBB. These drugs are administered in solvents such asethanol or DMSO and the dose of solvent is such thatBBB disruption may be caused by administration of thedrug/solvent mixture. Tween 80, also known as polysor-bate-80, is frequently administered in CNS drug formu-lations. A dose of polysorbate-80 of 3-30 mg/kg willcause BBB disruption in mice.30 Analgesia with kyotor-phin, a oligopeptide that normally does not cross theBBB, is possible following the peripheral administrationof the peptide, providing Tween 80 is coadministered.31

Low doses of another surfactant, SDS, are frequentlyincluded in CNS drug diluents. However, doses of SDSas low as 1.0 �g/kg can cause disruption of the BBB forshort periods. Immune adjuvants such as Freund’s com-plete or incomplete adjuvant cause disruption of the BBBto circulating IgG that can persist for weeks.32 This isrelevant to rodent vaccine models where active immuni-zation is attempted as a new therapy for the treatment ofbrain diseases. The vaccine for Alzheimer’s disease wasbased on the administration of the A� peptide mixed in

TABLE 1. BBB Disruption after Intracarotid Arterial Infusion of Noxious Agents

Method Comments (References)

Hyperosmolar Leads to chronic neuropathologic changes and vasculopathy in the brain and seizures21–25Vasoactive agents Examples are bradykinin, histamine, and multiple other vasoactive compounds; opens

BBB in brain tumor to greater extent than normal brain72Solvents BBB is solubilized with high dose ethanol, DMSO, SDS, Tween 80 (polysorbate-80)27–30Alkylating agents Examples are etoposide and melphalan; may alkylate key sulfhydryl residues similar to

mercury73,74Immune adjuvants Freunds adjuvant opens BBB to IgG for weeks; enable IgG uptake into brain in rodent

vaccine models, such as Alzheimer’s disease32Ultrasound The combination of administration of high-dose air bubbles (2–4 �m) and high-dose ul-

trasound (10–1000 watt/cm2) can induce BBB disruption75Cytokines Intracerebral interleukin-1� or CXC chemokines can attract white cells from blood and

cause BBB disruption76,77Miscellaneous Intracarotid acid pH, cold temperatures, or high-dose free fatty acid all cause BBB

disruption78–80



Freund’s adjuvant to transgenic mice with brain amy-loid.33 The adjuvant has two effects. First, it recruits theimmune system to the injection site so that antibodies aremade to the target peptide, in this case the A�. Second,the immune adjuvant causes an inflammatory responsethat results in opening of the BBB. This latter propertyallows the circulating anti-A� antibodies to enter thebrain. In the absence of BBB disruption, the circulatingIgG cannot enter the brain. In either active or passiveimmunization approaches to brain disorders, the circu-lating IgG must be enabled to cross the BBB and enterbrain to cause the intended pharmacological effect. IgGmolecules do not cross the BBB, in the absence of spe-cific transport mechanisms. It is unlikely that active orpassive immunization will be effective in humans, if theBBB is not disrupted.If a CNS drug is formulated in a vehicle other than a

physiological buffer, then the amounts of any solvent,surfactant, or adjuvant, that are included in the formula-tion should be evaluated critically as to whether drugtreatment is associated with solvent-mediated BBB dis-ruption. In this setting, there is a high likelihood thatchronic drug administration will have toxic side effects.

TRANS-NASAL DRUG DELIVERY TOTHE BRAIN

The delivery of drugs after intranasal administration isbased on the rationale that drugs can exit the submucousspace of the nose and cross the arachnoid membrane, andenter into olfactory CSF. It is posited that drug may thenenter the brain from the CSF flow tracts following intra-nasal administration of drug. There are two points toconsider when evaluating the potential efficacy of trans-nasal drug delivery to the brain. First, any drug thatenters into olfactory CSF will exit the CSF flow tractsand enter the peripheral bloodstream like any other ICVroute of administration. The second consideration is thatthe arachnoid membrane, which separates olfactory CSFfrom the submucous spaces of the nose, has high resis-tance tight junctions, just like the capillary endotheliumthat forms the BBB.34 Therefore, only lipid-soluble smallmolecules may cross the arachnoid membrane and enterinto olfactory CSF in the absence of arachnoid mem-brane disruption. Conversely, if the arachnoid membraneand other membranes in the nose are physically or chem-ically disrupted, then drug may enter the CSF from thenose. The human nasal cavity can only receive about 100�l per nostril without local injury.35 The volume of drugadministered into the nose is invariably ��200 �l. Mel-anocyte-stimulating hormone, a seven-amino acid neu-ropeptide, entered CSF following intranasal instillationin humans after these subjects ingested 20 consecutivepuffs of drug via an atomizer into each nares.36 Whendrug is administered to the nose via volumes that are not

injurious to the nose, then no distribution into CSF isfound for a water-soluble drug such as vitamin B12 or arelatively lipid soluble drug such as melatonin.35 In theabsence of local injury, distribution of neuropeptides toolfactory CSF is nil, unless the protein has access to aspecialized transport system that enables movementacross the arachnoid membrane. This was demonstratedin the case of a conjugate of HRP and wheat germagglutinin (WGA). The latter is a glycoprotein thatcrosses membranes via absorptive mediated endocytosis,based on binding to membrane lectin sites.37 Whereasthe HRP alone cannot penetrate the olfactory CSF, theHRP-WGA conjugate can cross plasma membranes viaabsorptive-mediated endocytosis.

TRANSVASCULAR DRUG DELIVERY TO THEBRAIN VIA ENDOGENOUS BBB

TRANSPORTERS

The complexity of the vascular tree in the cortex of ratbrain is shown with the India ink38 study in Figure 4. Thevascular density in the human brain is even more com-plex. In the human brain, there are over 100 billioncapillaries. The distance between capillaries is �50 �m.Therefore, the maximum diffusion distance in brain pa-renchyma following transvascular delivery is only 25�m. Even a molecule as large as albumin, 68,000 Damolecular mass, will diffuse 25 �m in less than 1 s.1

Because the intercapillary distance in brain is so small,every neuron is virtually perfused by its own blood ves-sel. The length of capillaries in human brain is �400miles, and the surface area of the brain capillary endo-thelium in the human brain is �20 m2. However, thevolume of the intraendothelial space is only 1 �l foradult rat brain and is only 5 ml for the human brain.Therefore, the brain capillary endothelial surface, whichforms the BBB in vivo, forms a very broad but thin

FIG. 4. India ink study shows vascular density in the cortex ofadult rat brain. Reprinted with permission from Bar. The vascularsystem of the cerebral cortex. Adv Anat Embryol Cell Biol 59:I–VI,1–62. Copyright © 1980, Springer-Verlag.38 All rights reserved.



barrier system. The thickness of the endothelial cell isonly �200 nm, which is less than 5% of the thickness ofmost cells.Transport across the BBB involves movement across

two membranes in series: the luminal and abluminalmembranes of the capillary endothelium, separated bythe 200 nm of endothelial cytoplasm. The microvascularendothelium in brain is completely invested by a base-ment membrane, but the basement membrane constitutesno diffusion barrier. Approximately 90% of the brainside of the capillary is covered by astrocyte foot process-es,39 although these astrocyte foot processes similarlyconstitute no diffusion barrier. Therefore, solutes freelyand instantaneously distribute throughout the entire brainextravascular volume after transport across the limitingmembrane, which is the capillary endothelial membrane.The BBB has a very high resistance owing to the tightjunctions, which cement adjacent endothelial cells to-gether. Due to the presence of the tight junctions, there isno para-cellular pathway for solute distribution intobrain interstitial fluid from blood. Circulating moleculescan only gain access to brain interstitium via a trans-cellular route through the brain capillary endothelialmembranes. If a molecule is lipid soluble and has amolecular mass less than 400 Da and is not avidly boundby plasma proteins or is a substrate for an active effluxtransport system at the BBB, then the circulating mole-cule may gain access to brain by lipid-mediated freediffusion. In the absence of the lipid-mediated pathway,circulating molecules may gain access to brain only viatransport on certain endogenous transport systems withinthe brain capillary endothelium. These endogenous trans-porters have an affinity for both small molecules andlarge molecules and can be broadly classified into threecategories: 1) CMT; 2) active efflux transport, or AET;and 3) receptor-mediated transport, or RMT.

CMTCMT systems for hexoses, monocarboxylic acids such

as lactic acid, neutral amino acids such as phenylalanine,basic amino acids such as arginine, quaternary ammo-nium molecules such as choline, purine nucleosides suchas adenosine, and purine bases such as adenine, areshown in Figure 5, which represents the luminal mem-brane of the brain capillary endothelium. The individualendogenous nutrients shown in Figure 5 are representa-tive substrates because each carrier system transports agroup of nutrients of common structure. The CMT sys-tems shown in Figure 5 are all members of the SoluteCarrier (SLC) gene family (Table 2). The BBB glucosecarrier is GLUT1 (glucose transporter type 1), which is amember of the SLC2 family; the BBB monocarboxylicacid transporter is MCT1, which is a member of theSLC16 family; the BBB large neutral amino acid andcationic amino acid transporters are LAT1 and CAT1,

respectively, which are members of the SLC7 family;LAT1 and CAT1 are the light chains of heterodimericproteins, and the heavy chain of the dimer is 4F2hc,which is a member of the SLC3 family; the BBB aden-osine transporter is CNT2, which is a member of theSLC28 family (Table 2). Each of the SLC familiesshown in Table 2 represent many common genes ofoverlapping nucleotide identity and some of the SLCfamilies are comprised of over 100 different genes.BBB GLUT1 transports glucose, 2-deoxyglucose,

3-O-methyl-glucose, galactose, and mannose, but not L-glucose.40 BBB MCT1 transports lactate, pyruvate, ke-tone bodies, and monocarboxylic acids.41 BBB LAT1transports the neutral amino acids with preferential af-finity for the large neutral amino acids.42 BBB CAT1transports arginine, lysine, ornithine.43 The BBB cholinetransporter transports choline, and perhaps other quater-nary ammonium molecules.44 To date, the BBB cholinetransporter has not been cloned. CHT1 is a sodium-dependent choline transporter member of the SLC5 fam-ily (Table 2), which corresponds to the sodium-depen-dent synaptosomal choline carrier. However, the BBBcholine transporter is sodium independent45 and is likelya member of a different SLC gene family. The BBBadenosine carrier transports adenosine, guanosine, andcertain pyrimidine nucleosides such as uridine,46 and isderived from the CNT2 gene,47 where CNT � concen-trative nucleoside transporter. Purine nucleosides arealso transported by sodium independent or equilibrativenucleoside transporters (ENT), which are members of theSLC29 gene family (Table 2). However, BBB transport

FIG. 5. BBB CMT systems are shown for seven different classesof nutrients, and the genes for five of these systems has beenidentified. GLUT1 � glucose transporter type 1; MCT1 � mono-carboxylic acid transporter type 1; LAT1 � large neutral aminoacid transporter type 1; CAT1 � cationic amino acid transportertype 1; CNT2 � concentrative nucleoside transporter type 2.



in vivo on the blood side of the endothelium is sodiumdependent,48 which excludes the role of an ENT carrierin mediating uptake of circulating adenosine. Pyrimidinenucleosides are primarily transported by CNT1, and, todate, there is no evidence that the BBB expresses CNT1.Purine bases such as adenine and guanine are transportedby a nucleobase transporter (NBT)46 but, to date, noeukaryotic NBT transporter gene has been cloned.In addition to the CMT systems shown in Figure 5,

there are many other CMT genes expressed at the BBB,which enable the BBB transport of water-soluble vita-mins, thyroid hormones, and other compounds. All of

these CMT systems at the BBB, which may number inthe dozens, are potential portals of entry of drugs to thebrain. The CMT systems comprise highly stereospecificpore-based transporters, and there are significant struc-tural requirements for transporter affinity. Therefore, it isunlikely that a drug, which is normally not transportedacross the BBB, would be made transportable by simplycoupling to the drug to another molecule that undergoesCMT across the BBB. Rather, the structure of the phar-maceutical should be altered with medicinal chemistry sothat it takes on the structure of a pseudo-nutrient and thusis able to undergo transport across the BBB via one of the

TABLE 2. Solute Carrier (SLC) Gene Families of Small-Molecule Transporters

Family Substrate Specificity Abbreviations

SLC1 Acidic amino acid transporter EEATASC small neutral amino acid transporter ASCT

SLC2 Glucose transporter GLUTH�-myo-inositol transporter HMIT

SLC3 Heavy chain of heterodimeric amino acid transporters 4F2hcSLC4 Bicarbonate/carbonate exchangers and Na� coupled transporters AE, NBCSLC5 Sodium/substrate cotransporters (glucose, choline) SGLT, CHTSLC6 Neurotransmitter transporters (GABA, glycine, taurine, monoamines, creatine) GAT, TAUTSLC7 Cationic amino acid transporter CAT

Light chain of amino acid transporters LATSLC8 Sodium/calcium exchanger NCXSLC9 Sodium/proton exchanger NHESLC10 Sodium/bile salt cotransporter NTCP, ASBTSLC11 Natural resistance-associated macrophage protein NRAMP

Divalent metal-ion transporter DMTSLC12 Potassium/chloride cotransporter KCCSLC13 Sodium/sulphate cotransporter NaS

Sodium/dicarboxylate transporter NaDCSLC14 Urea transporter UTSLC15 Proton peptide transporter PEPTSLC16 Monocarboxylic acid transporter (lactate, pyruvate, ketone bodies) MCTSLC17 Vesicular glutamic acid transporter VGLUTSLC18 Vesicular amine transporter VATSLC19 Vitamin transporters (folic acid, thiamine) THTRSLC20 Sodium-phosphate cotransporters PitSLC21 Organic anion transporters OATPSLC22 Organic cation transporters OCTN, OATSLC23 Sodium/ascorbic acid transporter SVCTSLC24 Sodium/calcium-potassium exchanger NCKXSLC25 Mitochondrial carriers MCSLC26 Anion exchangers CFTRSLC27 Fatty acid transport proteins FATPSLC28 Sodium dependent nucleoside transporters CNTSLC29 Equilibrative nucleoside transporters ENTSLC30 Zinc efflux transporters ZNTSLC31 Copper efflux transporters CTRSLC32 Vesicular neurotransmitter transporters VIAAT, VGATSLC33 Acetyl-CoA transporters ATSLC34 Sodium/phosphate cotransporters NaPiSLC35 Nucleotide sugar transporters UGTSLC36 Lysosomal amino acid transporters LYAATSLC37 Glucose-6-phosphate transporter G6PTSLC38 Sodium coupled neutral amino acid transporters SNATSLC39 Metal ion transporters ZIPSLC40 Iron efflux transporter MTP



CMT systems. For example, the �-carboxylation of do-pamine results in the formation of L-DOPA, and DOPA,a large neutral amino acid, is a substrate for the BBBLAT1. Once across the BBB, the L-DOPA is decarboxy-lated back to dopamine via aromatic amino acid decar-boxylase. L-DOPA is the primary example of a pro-drugthat traverses biological membranes, not via lipid medi-ation, but via carrier mediation.

AETP-glycoprotein is the prototypic AET system at the

BBB, and accounts for the active efflux of molecules inthe brain to blood direction. P-glycoprotein, which is aproduct of the ABC-B1 gene (FIG. 6), is just one ofmany members of the ATP binding cassette (ABC) genefamily of transporters. There are several multidrug resis-tance protein (MRP) transporters, which also belong tothe ABC gene family. The excessive focus on p-glyco-protein, also called the multidrug resistance (MDR) geneproduct, overlooks the fact that P-glycoprotein is just onemember of a large gene family, and many members ofthe ABC gene family may participate in BBB AET. Asecond consideration is that active efflux in the brain toblood direction requires the concerted actions of twodifferent types of transporters: an energy requiring trans-porter at one membrane of the endothelium, and an en-ergy-independent transporter, or exchanger, at the oppo-site membrane of the capillary endothelium. Examples ofenergy-independent exchangers are members of the sol-ute carrier (SLC) transporter gene family and include theorganic anion transporter (OAT) gene family or the or-ganic anion transporter polypeptide (OATP) gene family(FIG. 6). OATP and OAT are members of the SLC21and SLC22 gene families, respectively (Table 2).Certain drugs are excluded from penetration into brain

because these drugs are substrates for BBB AET sys-tems. One strategy for increasing brain penetration ofsuch drugs is the development of “co-drugs” that inhibitBBB AET systems and thereby allow increased brainpenetration of the therapeutic drug. The development ofpro-drugs to increase brain penetration of therapeuticsmight focus on MRP, OATP, or OAT transporters at theBBB in addition to p-glycoprotein.

RMTCertain large-molecule peptides or proteins undergo

transport from brain to blood via RMT across the BBB.There are at least three different types of BBB receptorsystems as depicted in Figure 7. The transferrin receptor(TfR) is an example of a bidirectional RMT system thatcauses both the receptor-mediated transcytosis of holo-transferrin in the blood to brain direction, and the reversetranscytosis of apo-transferrin in the brain to blood di-rection.49,50 The neonatal Fc receptor (FcRn) is an ex-ample of a reverse RMT system that functions only tomediate the reverse transcytosis of IgG in the brain toblood direction, but not in the blood to brain direc-tion.51,52 The type 1 scavenger receptor (SR-VI) is anexample of a receptor-mediated endocytosis system thatmediates the uptake of modified low-density lipoprotein(LDL) from the blood compartment into the intraendo-thelial compartment, and this endocytosis is not followedby exocytosis into brain interstitial fluid.53

Molecular Trojan horses and BBB RMTCertain endogenous ligands or peptidomimetic mAbs

that bind exofacial epitopes on BBB RMT systems andthat are endocytosing antibodies can act as molecularTrojan horses to ferry drugs, proteins, and nonviral genemedicines across the BBB using the endogenous RMT

FIG. 7. BBB RMT systems are shown for three classes ofsystems. An example of a bidirectional RMT system is the en-dothelial transferrin receptor (TfR), which mediates the transportof holo-transferrin (Tf) in the blood to brain direction, and thetransport of apo-Tf in the brain to blood direction. A reverse RMTsystem such as the neonatal Fc receptor (FcRn) transports IgGin the brain to blood direction only. An endocytosis system isillustrated by the type I scavenger receptor (SR-BI), which me-diates the endocytosis of acetylated low-density lipoprotein intothe endothelial compartment without transcytosis across theBBB.

FIG. 6. BBB AET systems are comprised of an energy-depen-dent system at one side of the brain capillary endothelium andan energy-independent system at the opposite endothelialmembrane. As a hypothetical example, members of the ABCgene family are shown at the luminal endothelial membrane, andmembers of the SLC gene family are shown at the abluminalendothelial membrane.



systems. This BBB molecular Trojan horse technologyhas been reduced to practice in vivo in the followingsystems:

• Vasoactive intestinal peptide (VIP) causes a 60%increase in cerebral blood flow after intravenousinjection in conscious rats.54

• BDNF causes 100% normalization of the pyramidalcell density in the CA1 sector of the hippocampusin adult rats subjected to transient forebrain isch-emia after delayed intravenous administration.55

• BDNF reduces stroke volume 65-70% in adult ratswith either permanent or reversible middle cerebralartery occlusion (MCAO) after delayed intravenousadministration.56,57

• FGF-2 causes an 80% reduction in stroke volume ina permanent MCAO model in adult rats after de-layed intravenous administration.58

• Epidermal growth factor (EGF) can be used as apeptide radiopharmaceutical to enable early detec-tion of brain cancer that overexpresses the EGFreceptor.59

• A�1-40 can be used as a peptide radiopharmaceuti-cal for the early detection of brain amyloid in Alz-heimer’s disease.60

• Sequence-specific peptide nucleic acids (PNA) canbe used as antisense radiopharmaceuticals for the invivo imaging of gene expression in brain, in eithertransgenic mouse models or adult rats with exper-imental brain cancer.61,62

In all of these studies, the peptide or antisense agentwas ineffective in the brain in vivo after intravenousadministration owing to the lack of transport of the mol-ecule across the BBB. However, the intended CNS phar-macologic effect in vivo was achieved after intravenousadministration, owing to conjugation of the peptide orantisense therapeutic to a BBB molecular Trojan horse.Molecular Trojan horses can also target liposomes63 andnanoparticles64 across the BBB. Nonviral plasmid DNAis encapsulated in pegylated liposomes, which are thentargeted across the BBB and the brain cell membranewith peptidomimetic monoclonal antibodies that func-tion as molecular Trojan horses.65 The pegylated immu-noliposome (PIL) nonviral gene transfer technology hasenabled 100% normalization of striatal tyrosine hydrox-ylase activity in experimental Parkinson’s,66 and a 100%increase in survival time of adult mice with experimentalbrain cancer.67 After intravenous administration of PILscarrying an exogenous reporter gene, the exogenous genewas globally expressed in all regions of the brain of theadult Rhesus monkey after intravenous injection of anonviral formulation.68 Plasmid DNA that producesshort hairpin RNA for the purposes of silencing genes

through a mechanism of RNA interference (RNAi) canbe delivered across the BBB with the PIL gene targetingtechnology.69 This resulted in an 88% increase in sur-vival time in adult mice with experimental human braincancer that were treated with DNA-based RNAi thera-peutics directed against the human EGF receptor.70

CONCLUSIONS

The development of new drugs for brain disorders is aformidable challenge, and there is no effective treatmentfor the majority of brain diseases (FIG. 2). The inabilityto treat most brain diseases is incongruous with the tre-mendous progress made in the molecular neurosciences.The brain drug discovery sciences have, in fact, beenhighly successful, and many new therapeutics have beendiscovered, which could potentially be used to treat thebrain, if the BBB problem was solved. However, if thedrugs cannot be delivered across the BBB, then there isno translation from the lab to the clinic. Step number 1 inCNS drug development is providing solutions to theBBB problem (FIG. 8). If no BBB delivery solutions arein place, which is the standard in the pharmaceuticalindustry, then the number of drugs that can be developedas new neurotherapeutics is less than 2% of small mol-ecules and is �0% of large molecules. The few smallmolecules that do cross the BBB are those drugs thathave high lipid solubility and molecular mass less than400 Da, and these drugs generally only treat certain CNSdisorders, such as epilepsy, affective disorders, andchronic pain (FIG. 8). In the absence of an effective BBBtechnology, the pharmaceutical industry cannot providetherapeutics for the majority of patients with brain dis-orders. It is estimated that the global CNS pharmaceuti-cal market would have to grow by more than 500% justto equal the cardiovascular market,71 and there are more

FIG. 8. Step 1 in CNS drug development is the availability ofeffective BBB drug or gene targeting technology. In the absenceof a BBB technology, then the CNS drug developer is limited tolipid-soluble low molecular weight drugs, and only a few CNSdiseases consistently respond to this class of molecule.



patients with CNS disorders than there are with cardio-vascular disease. If BBB delivery solutions were in placefor either small or large molecules, then almost anypharmaceutical could enter clinical drug developmentprograms and therapies could be developed for mostCNS disorders (FIG. 8).

Acknowledgments: This work was supported by NationalInstitutes of Health Grant NS34698.

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